Repression via Facultative Heterochomatin

描述（由申请人提供）：本提案研究表观遗传学的分子方面。表观遗传学包括基因表达谱的变化，而不改变细胞的基因组DNA序列。这源于真核生物通过一系列压实来构造染色体DNA区域的动态过程。基因表达模式的改变对细胞分化和发育至关重要，并由子细胞遗传，从而维持给定细胞类型的完整性、规格和功能。这种表观过程中的异常会引起扰动，这种扰动也会遗传并破坏正常的细胞特性。将DNA包装成染色质的组蛋白受到翻译后修饰的影响，从而产生不同的染色质结构。虽然常染色质具有允许转录的宽松结构，但组成型异染色质密集排列且难以接近。本提案的重点是兼性染色质，它是压抑的，但可以改变其性质并变得活跃。目的是确定控制兼性异染色质形成的分子机制和确保其通过细胞分裂繁殖的表观遗传参数。我们专注于影响兼性异染色质形成的三个主要活动：SirT1， L3MBTL1 （L1）和L3MBTL2 (L2)，它们组成了定义的多蛋白复合物，通过这些复合物促进了它们的作用。SirT1是一种NAD+组蛋白去乙酰化酶，在体内靶向组蛋白h4 -赖氨酸16 （H4K16）去乙酰化，并与组蛋白H1b和组蛋白赖氨酸甲基转移酶Suv39h1相互作用。L1和L2都含有MBT结构域，与核小体结合并与HP1 γ相关。L1还与组蛋白H1结合，并结合两个抑制性组蛋白标记。L2与三个无名指蛋白和E2F6结合，催化组蛋白H2AK119的单泛素化。因此，SirT1、L1和L2都在与兼性异染色质相关的组蛋白残基上表现出活性。我们将确定每个基因的靶基因（全基因组ChIP on ChIP实验），它们在靶基因上的定位作为基因表达的功能，作为使用高度特异性抗体的特定组蛋白修饰的功能（免疫荧光和ChIP分析），以及组蛋白H1和其他相互作用因子在染色质压实分析中的作用（包括蔗糖梯度沉降和电子显微镜）。

DESCRIPTION (provided by applicant): This proposal investigates molecular aspects of epigenetics. Epigenetics encompasses changes in gene expression profiles that occur without alterations in the genomic DNA sequence of a cell. This arises from the dynamic processes that structure regions of chromosomal DNA through a range of compaction in eukaryotes. The altered pattern of gene expression is pivotal to cellular differentiation and development and is inherited by daughter cells thereby maintaining the integrity, specifications, and functions for a given cell type. Aberrancies in this epigentic process gives rise to perturbations that are also inherited and disruptive to normal cellular properties. The histone proteins that package DNA into chromatin are subject to post- translational modifications that give rise to different chromatin structures. While euchromatin has a relaxed structure permissive to transcription, constitutive heterochromatin is densely packed and inaccessible. The focus of this proposal is facultative chromatin that is repressive, but can be altered in its properties and become active. The aims are to identify the molecular mechanisms controling the formation of facultative heterochromatin and the epigenetic parameters that ensure its propagation through cell divisions. We focus on three major activities that impact facultative heterochromatin formation: SirT1, L3MBTL1 (L1) and L3MBTL2 (L2) that comprise defined multiprotein complexes through which their role is facilitated. SirT1 is a NAD+ histone deacetylase that targets histone H4-lysine 16 (H4K16) for deacetylation in vivo, and interacts with histone H1b and the histone lysine methyltransferase Suv39h1. Both L1 and L2 contain MBT domains, bind to nucleosomes and associate with HP1 gamma. L1 also associates with histone H1 and binds to two repressive histone marks. L2 associates with three ring finger proteins and E2F6 and catalyzes monoubiquitination of histone H2AK119. Thus all three, SirT1, L1, and L2 exhibit activites on histone residues associated with facultative heterochromatin. We will identify the target genes for each (genome- wide ChIP on ChIP experiments), their localization on target genes as a function of gene expression and as a function of specific histone modifications using highly specific antibodies (immunofluoresecnce and ChIP analyses), and the role of histone H1 and other interacting factors in chromatin compaction analyses (including sucrose gradient sedimentation together with electron microscopy).